A Mass Spectrometer is an analytical instrument that can separate charged molecules according to their masstocharge ratio.

The mass spectrometer can answer the questions what is in the sample (qualitative structural information) and how much is present (quantitative determination) for a very wide range of samples at high sensitivity

3Components of a Mass SpectrometerVacuum SystemAtmosphere 4How are mass spectra produced ?

Ions are produced in the source and are transferred into the mass analyser

They are separated according to their mass/charge ratio in the mass analyser (e.g. Quadrupole, Ion Trap)

Ions of the various m/z values exit the analyser and are counted by the detector

5What is a Mass Spectrum ?

A mass spectrum is the relative abundance of ions of different m/z produced in an ion source

-a chemical fingerprint

It contains

Molecular weight information (generally)

Structural Information (mostly)

Quantitative information

6What does a mass spectrum look like ?

Electrospray mass spectrum of salbutamol

240100Base peak at m/z 240

24106080100120140160180200220240260280300 7What information do you need from the analysis ?

Low or High Mass range

Average or Monoisotopic mass (empirical)

Accurate Mass

Quantitation - precision, accuracy, selectivity

Identification

Structural Information

Isotope Ratios

8Mass Analyzers

Types of Mass Spectrometer

Magnetic Sector

Quadrupole

Ion Trap

Time-of-flight

Hybrid- Sector/trap, Quad/TOF etc

Mass Spectrometers separate ions according to their mass-to-charge (m/z) ratios

9A Triple Stage Quadrupole Mass AnalyzerFinnigan TSQQ2 is Non-Linear Collision CellQ0Q1Q2Q3ESI ProbeSquare Rod Ion Transmission to Analytical QuadsElectron Multiplier, Detection SystemHyperbolic, high precision quadrupoles 10Hyperbolic Quadrupoles 11How does the quadrupole work ?The quadrupole consists of four parallel rods. The opposing rods have the same polarity whilst adjacent rods have opposite polarity.Each rod is applied with a DC and an RF voltage.Ions are scanned by varying the DC/Rf quadrupole voltages. Only ions with the selected mass to charge ratio will have the correct oscillatory pathway in the Rf field. 12Quadrupole Mass Analyser

The ion is transmitted along the quadrupole in a stable trajectory Rf field. The ion does not have a stable trajectory and is ejected from the quadrupole.

13Non-linear collision cell in the TSQA non-linear Q2 ensures that neutral species are ejected from the mass analyser pathway 14Non-linear collision cell in TSQNon-linear collision cell removes the line of sight to the detector for neutral species - reducing the noise at the detector. 15A 90 Degree Square Q2 in the TSQ QuantumAPI 16Ion Trap

Consists of ring electrode and two end caps

Principle very similar to quadrupole

Ions stored by RF DC fields

Scanning field can eject ions of specific m/z

Advantages

- MS/MS/MS..

- High sensitivity full scan MS/MS

17Resolving Power of a quadrupoleThe ability of a quadrupole to resolve masses is proportional to frequency of AC and length of quadrupole 18Resolving power of round rodsRound rods can only approximate the field generated by hyperbolic rods - most ideal when Rrod/R0 1.16Rrod Radius of rod R0 Position of rod relative to axis of instrument 19Resolving power of round rodsManufacturing rods such that Rrod/R0 1.16 over the full length of quadrupole is non-trivial. Variability would assure that one M is selected at one position and another where the ratio is different - loss of specificity and sensitivity. Thermal expansion coefficients must be very low expansion / contraction within the length of the rod changes Rrod/R0 Hyperbolic rods give excellent transmission across the full range of resolution - providing optimal analytical specificity. Compare sensitivity at unit resolution versus open resolution. At tighter resolution round rods systems tend to lose a greater proportion of ions through variation in Rrod/R0. 20Manufacture of round rodsRound rods manufacture typically requires several steps - first, machine rods. Second, machine holder by drilling four holes into a cylinder. Third, machine dropout to create ion path. Insert rods. Error in Rrod/R0 grows by number of processes. 21Resolution BasicsMagnetic Sector Instruments Constant Resolution with mass 10 Valley DefinitionResolution m / ? m 22Resolution BasicsQuadrupoles Ion traps and TOFs Constant peak widths Variable Resolution FWHM DefinitionResolution m / ? mm/z 500/1, R 500 m/z 500/0.1, R 5000 23What is the advantage of using high resolution mass spectrometry?The advantage of high resolution measurement is to eliminate chemical background of the same nominal mass but different accurate mass and, therefore to increase the signal to noise ratio and the sensitivity respectively. Better selectivity is obtained by applying high resolution in case of isobaric compounds, i.e. two compounds of same nominal mass but different accurate mass. With low resolution only a combined Spectral result is obtained under Product ion conditions . With high resolution separate detection and therefore separate isolation and MS/MS spectra are obtained. Example R 1000 R 5000 Compound mass1 372.351 Da OVERLAPPING SEPARATION Compound mass2 372.421 Da 24Effect of changing resolution on peak shape0.5 FWHM1.0 FWHM0.7 FWHM0.3 FWHM0.2 FWHM0.1 FWHM 25Effect of Peak Width On TransmissionQUADRUPOLEROUND RODS100 Resolution at m/z 500Tr80Unitan1000 60s2500

gt5000 (FWHM) parent ion resolution allows mass selection of these compounds differing by less than 0.2 Da from PPG

Increasing the resolution to exclude the PPG interference increases the selectivity and specificity for the quantitative assay

27Mometasone resolution in the presence of PPGs (Mometasone MH 521.2, PPG (n8) MNa 521.3) 28Mometasone resolution in the presence of PPGsproduct ion spectra of 37Cl mometasone at various precursor ion resolutions PPG - related peaksQ1 resolution unitQ1 resolution 0.08 FWHM passing only mometasone 37ClThe increased resolution on product ion scan of 37Cl mometasone can selectively exclude PPG interferents with an actual mass difference of less than 0.2 Da. 29Alprazolam resolution in the presence of PPGs(alprazolam MH 309.0907, PPG (n5) MH 309.2277)250 pg/uL alprazolam in 20 uM PPGs 30Alprazolam resolution in the presence of PPGs Product ion spectra at various precursor ion resolutionsQ1 set at unit resolution, spectrum is a composite of alprazolam and interfering PPG ionsQ1 set to pass only PPG ionsQ1 set to pass only alprazolam ionsThe increased resolution on product ion scan of alprazolam can selectively exclude PPG interferents with an actual mass difference of less than 0.2 Da. 31Comparison of Quads and Traps

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